CN113151314B - Plant ACCase mutant gene and application thereof - Google Patents
Plant ACCase mutant gene and application thereof Download PDFInfo
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- CN113151314B CN113151314B CN202110506569.5A CN202110506569A CN113151314B CN 113151314 B CN113151314 B CN 113151314B CN 202110506569 A CN202110506569 A CN 202110506569A CN 113151314 B CN113151314 B CN 113151314B
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/93—Ligases (6)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y604/00—Ligases forming carbon-carbon bonds (6.4)
- C12Y604/01—Ligases forming carbon-carbon bonds (6.4.1)
- C12Y604/01002—Acetyl-CoA carboxylase (6.4.1.2)
Abstract
The invention discloses a plant herbicide-resistant ACCase mutant gene, wherein the plant ACCase gene is subjected to the following two mutations because of a wild ACCase gene: the 5779 th and 5780 th site nucleotides are mutated from C to T and C to A, respectively, or the 5779 th, 5780 th and 5781 th site nucleotides are mutated from C to T, C to A and T to C, respectively. The inventors of the present application found several mutant genes, corresponding to plants having resistance (tolerance) to herbicides of the acetyl-coa carboxylase inhibitor class, and confirmed that they have very good resistance (tolerance) to herbicides when applied to rice.
Description
Technical Field
The invention belongs to the field of plant genetic engineering and plant herbicide-resistant breeding, and relates to creation of an endogenous ACCase herbicide-resistant locus of a plant.
Background
With the large-area application of the direct seeding cultivation technology of plants in agricultural production, the problem of weeds in the farmland in the direct seeding production of plants becomes an important factor influencing the crop yield. In particular, the physiological and metabolic processes of the gramineous weeds in the rice field are highly similar to those of plants, and proper herbicides are lacked for prevention and control. Because the traditional breeding and domesticating process does not relate to herbicide tolerance characters, resistance resources in natural genetic resources are extremely rare. At present, the herbicide resistance character widely used in plants is obtained by expressing heterologous metabolic genes through a transgenic means, and the herbicide resistance character is difficult to apply to main grain crops such as plants and the like in a short period. The method is a feasible way for developing the herbicide-resistant germplasm resources of the main grain crops at present by utilizing the herbicide inhibition target gene and screening and identifying the endogenous resistance mutants. For example, resistance resources can be obtained by mutating the imidazole herbicide target gene ALS by EMS. In recent years, the mutation is widely applied to varieties such as 'Jinjing 818' and the like, and shows remarkable cultivation advantages in the aspect of chemical weeding. However, it is known that endogenous resistance sites of major herbicide target genes such as EPSPS, ACCase, ALS, HPPD, etc. are mostly patented by foreign commercial companies. At the same time, field weed acquired resistance rapidly increases with repeated application of a single type of herbicide. Therefore, the discovery of new sites of novel herbicide resistance genes of plants is necessary for the cultivation of herbicide resistant plants.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide a plant mutant gene which can impart resistance to (tolerance to) acetyl-coa carboxylase herbicides to plants.
To this end, the invention provides a nucleic acid or gene encoding a plant ACCase mutant encoding said protein.
The nucleic acid or gene of the present invention comprises:
on the basis of the wild-type ACCase gene, there are two mutated genes: the nucleotide at the 5779 and 5780 positions is mutated, or the nucleotide at the 5779, 5780 and 5781 positions is mutated.
Preferably, the plant ACCase mutant gene of the present invention has the following two types of mutations: the 5779 th and 5780 th nucleotides are mutated from C to T and C to A, respectively, or the 5779 th, 5780 th and 5781 th nucleotides are mutated from C to T, C to A and T to C, respectively.
The nucleotide mutated in the ACCase mutant gene of the present invention is also within the scope of the present invention when the mutation is made at the above-mentioned position to another nucleotide, for example: the C mutation is A, T and G, or the T mutation is A, C and G.
Preferably, the nucleotide sequence of the ACCase mutant gene comprises a sequence shown in SEQ ID NO. 1 of the sequence Listing.
The invention also provides an expression cassette or a recombinant vector, which contains the nucleic acid or the gene.
The invention also provides application of the plant expression cassette and the recombinant vector in the aspect of herbicide resistance of plants, and the plant expression cassette and the recombinant vector are used for improving the herbicide resistance of target plants.
The present invention also provides a method for obtaining a plant having herbicide resistance, comprising the steps of:
1) Allowing the plant to comprise said nucleic acid or gene.
Further, the method of obtaining a plant with herbicide resistance is to utilize CRISPR-mediated guided editing pathway.
The invention also provides methods of obtaining plants that are herbicide resistant. Further, the present invention provides methods for enhancing the tolerance of a plant, plant tissue, plant cell to at least one herbicide that has an activity that interferes with the ACCase enzyme. Thus, herbicide resistance in the present invention refers to resistance to (tolerance to) acetyl-coa carboxylase herbicides.
The method of enhancing the resistance of a plant, plant tissue or plant cell to a herbicide of the present invention can be carried out by transformation or crossing, selfing and asexual propagation, or by site-directed mutagenesis of a gene, such that the altered plant comprises the nucleotide sequence of SEQ ID No. 1 of the present invention.
The method of enhancing the resistance of a plant, plant tissue or plant cell to a herbicide according to the present invention can be carried out by transformation or by crossing, selfing and asexual propagation.
The invention also provides a herbicide-tolerant plant, which expresses an ACCase gene containing a herbicide-resistant mutation site, wherein the gene sequence is different from that of a wild-type plant and is a sequence of the ACCase gene containing the herbicide-resistant mutation site. Plants carrying ACCase mutation sites were found to be resistant to ACCase herbicides, whereas wild type plants appeared to be sensitive to ACCase herbicides. The invention also provides the application of the nucleic acid or the gene and the protein in plant breeding, which is used for cultivating plants with herbicide resistance, in particular crops, and also provides the application of the protein and the coding gene thereof in transgenic or non-transgenic plants such as rice and the like.
Drawings
FIG. 1 shows the comparison of herbicide resistant rice plants containing ACCase mutation sites with the corresponding wild type.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the embodiments of the present invention are not limited thereto.
The present invention will be described below with reference to examples. It will be understood by those skilled in the art that the following examples are illustrative only and are not intended to limit the scope of the present invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. The raw materials and reagents used in the following examples are all commercially available products unless otherwise specified.
Example 1 plant ACCase mutation site acquisition and construction of plant expression vectors
Taking rice as an example, the nucleotide sequence in the ACC gene of rice was selectedCCAGCCTATTATTCTTACAGGCT (the underlined part is the PAM sequence of the 5'NGG-3' structure) as the targeting site. Synthesizing (general biology, ltd.) a forward oligonucleotide chain (ACC-PE FP) and a complementary reverse oligonucleotide chain (ACC-PE RP) according to the selected target sites,
the specific sequence is as follows:
ACC-PE FP1:GGCAagcctgtaagaataataggcGTTTC
ACC-PE FP1:CTCTGAAACgcctattattcttacaggct
ACC-PE FP2:GTGCtgaccagNNNattattcttacag
ACC-PE RP2:AAAActgtaagaataatNNNctggtca
and respectively annealing two chains of ACC-PE FP1+ RP1 and ACC-PE FP2+ RP2 to form duplex DNA with sticky ends through an annealing program, wherein the duplex DNA is used as an insert fragment for constructing a recombinant vector.
And performing guided editing by using a CRISPR (clustered regularly interspaced short palindromic repeats) -mediated guided editing system. Specifically, the SpCas9 protein-mediated guide editing system pHUN411-PE2, which can introduce pre-designed mutations on the genome, was used.
The pHUN411-PE2 vector was digested with BsaI endonuclease (NEB) at 37 ℃ for 4 hours, and the digestion system was inactivated at 65 ℃ for 10 minutes, to serve as a backbone fragment for constructing a recombinant vector. The recombinant vector backbone fragment and insert, and sgRNA Scoffold were ligated end-to-end by the Goldgate method (NEB) and introduced into E.coli. Positive transformants were obtained by selecting plaques with kanamycin resistance and no spectinomycin resistance. After sequencing verification, positive plasmids are extracted to form a recombinant vector plasmid for rice ACC gene CRISPR/Cas9 targeting, which is named as pHUN411-PE2-ACC PegRNA, a plant expression vector is transferred into Agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105 (plant group preservation of the center for component supervision and inspection of transgenic biological products of the department of agriculture of the academy of agricultural sciences of Anhui) by a freeze-thaw method, and positive clones are obtained by colony PCR screening, so that the Agrobacterium containing the pHUN411-PE2-ACC PegRNA is obtained.
Example 2 Positive transgenic plants
After the glumes of the mature rice seeds are removed, the seeds are soaked in 70% alcohol for 1min, and the alcohol is poured off. Seeds were soaked for 40min (150 r/min) with 1 drop of Tween 20 in 50% sodium hypochlorite (stock solution available chlorine concentration greater than 4%). The sodium hypochlorite is poured off, and the solution is washed for 5 times by sterile water until the solution is clear until the taste of the sodium hypochlorite does not exist. The seeds were soaked in sterile water overnight. The embryos were peeled off with a scalpel along the aleurone layer of the seeds and inoculated on callus induction medium. After dark culture for 11 days at 30 ℃, separating the callus from endosperm and embryo, and pre-culturing the primary callus with good bud removing state and vigorous division for 3-5 days for agrobacterium transformation.
Agrobacterium tumefaciens transformed with the recombinant expression vector in the above-described procedure is used to perform Agrobacterium-mediated genetic transformation, and positive plants are obtained by the methods proposed by Yongbo Duan (Yongbo Duan, chenguang Zhai, et al. An infection and high-throughput protocol for Agrobacterium mediated transformation based on phosphorus hormone mutant mutation selection in Japonica rice (Oryza sativa L.) [ J. Plant Report,2012.DOI10.1007/s 00299-012-1275-3.) and the like.
The corresponding callus of the wild type was cultured in a similar manner to obtain a control plant.
Example 3: mutant site analysis of plant acetyl coenzyme A carboxylase herbicide resistant mutant
And selecting leaves of the obtained herbicide-resistant rice mutant plants, extracting genome DNA, and sending the genome DNA to Invitrogen company for genome sequencing. The sequencing result is compared with the wild type Nipponbare ACCase gene, and the nucleotide sequence of the ACCase gene of 2 or 3 sites of mutation, the base of the 5779, 5780 and 5781 sites of mutation, respectively from C to T and C to A, or the nucleotide of the 5779, 5780 and 5781 sites of mutation, respectively from C to T, C to A and T to C, namely herbicide resistant mutant, is shown as SEQ ID NO. 1 in the sequence list.
The mutant plants still showed resistance to the herbicide after being sprayed with 0.15g/L haloxyfop 2 times every 3 days, whether in a 2 μ M haloxyfop herbicide-resistant medium or transplanted into soil, as compared with wild type plants (FIG. 1).
While the principles of the invention have been described in detail in connection with the preferred embodiments thereof, it will be understood by those skilled in the art that the foregoing embodiments are merely illustrative of exemplary implementations of the invention and are not limiting of the scope of the invention. The details in the examples are not to be construed as limitations on the scope of the invention, and any obvious modifications, equivalent alterations, simple substitutions, etc. based on the technical solution of the present invention are intended to fall within the scope of the present invention without departing from the spirit and scope of the present invention.
Sequence listing
<110> institute of Paddy Rice of agricultural science institute of Anhui province
<120> plant ACCase mutant gene and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 7246
<212> DNA
<213> mutant Gene (ACCase)
<400> 1
atgccgatgc ggccgtggga atttattatt ttaggccgca cagcccccat ctctccccca 60
cccctcggga tatccgcctt ctcccgcccg ctccgcctcc gcctccgcct ccgcctcgcc 120
gccgttgtcg acgccgcaag gatccaaacg ccgccccgcg tcgccttctc cgccttcttc 180
ttcttcttct tcttcttctc cgagggtctc tctcgggaag gtacagctgc ccgcctccct 240
gctcttattt atcgcgtgcc gttcattccg tgccttctcc agctccagat ccgcgcgccg 300
cacgccgatc cgcgcccggc cggttttagc tgcgctcatc atttcctcgc gttggctcag 360
ggtttaagct cctctttgtt aagtgggacg atttcacaca tctggggatt tatcttctct 420
ttgtatggca ctacacattt gagaaaccgt gcaattctac tgtttggtct tgctggcatc 480
attgacctcc caaatgacgc agcttcagaa gttgatattt cacatggttc cgaagatccc 540
agggggccta cggtcccagg ttcctaccaa atgaatggga ttatcaatga aacacataat 600
gggaggcatg cttcagtctc caaggttgtt gagttttgta cggcacttgg tggcaaaaca 660
ccaattcaca gtgtattagt ggccaacaat ggaatggcag cagctaagtt catgcggagt 720
gtccgaacat gggctaatga tacttttgga tcagagaagg caattcagct gatagctatg 780
gcaactccgg aggatctgag gataaatgca gagcacatca gaattgccga tcaatttgta 840
gaggtacctg gtggaacaaa caacaacaac tatgcaaatg tccaactcat agtggagata 900
gcagagagaa caggtgtttc tgctgtttgg cctggttggg gtcatgcatc tgagaatcct 960
gaacttccag atgcgctgac tgcaaaagga attgtttttc ttgggccacc agcatcatca 1020
atgcatgcat taggagacaa ggttggctca gctctcattg ctcaagcagc tggagttcca 1080
acacttgctt ggagtggatc acatgtggaa gttcctctgg agtgttgctt ggactcaata 1140
cctgatgaga tgtatagaaa agcttgtgtt actaccacag aggaagcagt tgcaagttgt 1200
caggtggttg gttatcctgc catgattaag gcatcttggg gtggtggtgg taaaggaata 1260
aggaaggttc ataatgatga tgaggttagg acattattta agcaagttca aggcgaagta 1320
cctggttccc caatatttat catgaggcta gctgctcaga gtcgacatct tgaagttcag 1380
ttgctttgtg atcaatatgg caacgtagca gcacttcaca gtcgagattg cagtgtacaa 1440
cggcgacacc aaaagataat cgaggaagga ccagttactg ttgctcctcg tgagactgtg 1500
aaagagcttg agcaggcagc acggaggctt gctaaagctg tgggttatgt tggtgctgct 1560
actgttgaat acctttacag catggaaact ggtgaatatt attttctgga acttaatcca 1620
cggctacagg ttgagcatcc tgtcactgag tggatagctg aagtaaattt gcctgcggct 1680
caagttgctg ttggaatggg tatacccctt tggcagattc cagagatcag gcgcttctac 1740
ggaatgaacc atggaggagg ctatgacctt tggaggaaaa cagcagctct agcgactcca 1800
tttaactttg atgaagtaga ttctaaatgg ccaaaaggcc actgcgtagc tgttagaata 1860
actagcgagg atccagatga tgggtttaag cctactggtg gaaaagtaaa ggagataagt 1920
ttcaagagta aaccaaatgt ttgggcctat ttctcagtaa agtctggtgg aggcatccat 1980
gaattcgctg attctcagtt cggacatgtt tttgcgtatg gaactactag atcggcagca 2040
ataactacca tggctcttgc actaaaagag gttcaaattc gtggagaaat tcattcaaac 2100
gtagactaca cagttgacct attaaatgcc tcagatttta gagaaaataa gattcatact 2160
ggttggctgg ataccaggat agccatgcgt gttcaagctg agaggcctcc atggtatatt 2220
tcagtcgttg gaggggcttt atataaaaca gtaactgcca acacggccac tgtttctgat 2280
tatgttggtt atcttaccaa gggccagatt ccaccaaagc atatatccct tgtctatacg 2340
actgttgctt tgaatataga tgggaaaaaa tatacaatcg atactgtgag gagtggacat 2400
ggtagctaca gattgcgaat gaatggatca acggttgacg caaatgtaca aatattatgt 2460
gatggtgggc ttttaatgca gctggatgga aacagccatg taatttatgc tgaagaagag 2520
gccagtggta cacgacttct tattgatgga aagacatgca tgttacagaa tgaccatgac 2580
ccatcaaagt tattagctga gacaccatgc aaacttcttc gtttcttggt tgctgatggt 2640
gctcatgttg atgctgatgt accatatgcg gaagttgagg ttatgaagat gtgcatgccc 2700
ctcttatcac ccgcttctgg tgtcatacat gttgtaatgt ctgagggcca agcaatgcag 2760
gctggtgatc ttatagctag gctggatctt gatgaccctt ctgctgttaa gagagctgag 2820
ccgttcgaag atacttttcc acaaatgggt ctccctattg ctgcttctgg ccaagttcac 2880
aaattatgtg ctgcaagtct gaatgcttgt cgaatgatcc ttgcggggta tgagcatgat 2940
attgacaagg ttgtgccaga gttggtatac tgcctagaca ctccggagct tcctttcctg 3000
cagtgggagg agcttatgtc tgttttagca actagacttc caagaaatct taaaagtgag 3060
ttggagggca aatatgagga atacaaagta aaatttgact ctgggataat caatgatttc 3120
cctgccaata tgctacgagt gataattgag gaaaatcttg catgtggttc tgagaaggag 3180
aaggctacaa atgagaggct tgttgagcct cttatgagcc tactgaagtc atatgagggt 3240
gggagagaaa gtcatgctca ctttgttgtc aagtcccttt ttgaggagta tctctatgtt 3300
gaagaattgt tcagtgatgg aattcagtct gatgtgattg agcgtctgcg ccttcaacat 3360
agtaaagacc tacagaaggt cgtagacatt gtgttgtccc accagagtgt tagaaataaa 3420
actaagctga tactaaaact catggagagt ctggtctatc caaatcctgc tgcctacagg 3480
gatcaattga ttcgcttttc ttcccttaat cacaaagcgt attacaagtt ggcacttaaa 3540
gctagtgaac ttcttgaaca aacaaaactt agtgagctcc gtgcaagaat agcaaggagc 3600
ctttcagagc tggagatgtt tactgaggaa agcaagggtc tctccatgca taagcgagaa 3660
attgccatta aggagagcat ggaagattta gtcactgctc cactgccagt tgaagatgcg 3720
ctcatttctt tatttgattg tagtgataca actgttcaac agagagtgat tgagacttat 3780
atagctcgat tataccagcc tcatcttgta aaggacagta tcaaaatgaa atggatagaa 3840
tcgggtgtta ttgctttatg ggaatttcct gaagggcatt ttgatgcaag aaatggagga 3900
gcggttcttg gtgacaaaag atggggtgcc atggtcattg tcaagtctct tgaatcactt 3960
tcaatggcca ttagatttgc actaaaggag acatcacact acactagctc tgagggcaat 4020
atgatgcata ttgctttgtt gggtgctgat aataagatgc atataattca agaaagtggt 4080
gatgatgctg acagaatagc caaacttccc ttgatactaa aggataatgt aaccgatctg 4140
catgcctctg gtgtgaaaac aataagtttc attgttcaaa gagatgaagc acggatgaca 4200
atgcgtcgta ccttcctttg gtctgatgaa aagctttctt atgaggaaga gccaattctc 4260
cggcatgtgg aacctcctct ttctgcactt cttgagttgg acaagttgaa agtgaaagga 4320
tacaatgaaa tgaagtatac cccatcacgg gatcgtcaat ggcatatcta cacacttaga 4380
aatactgaaa accccaaaat gttgcaccgg gtatttttcc gaacccttgt caggcaaccc 4440
agtgtatcca acaagttttc ttcgggccag attggtgaca tggaagttgg gagtgctgaa 4500
gaacctctgt catttacatc aaccagcata ttaagatctt tgatgactgc tatagaggaa 4560
ttggagcttc acgcaattag aactggccat tcacacatgt atttgcatgt attgaaagaa 4620
caaaagcttc ttgatcttgt tccagtttca gggaatacag ttttggatgt tggtcaagat 4680
gaagctactg catattcact tttaaaagaa atggctatga agatacatga acttgttggt 4740
gcaagaatgc accatctttc tgtatgccaa tgggaagtga aacttaagtt ggactgcgat 4800
ggtcctgcca gtggtacctg gaggattgta acaaccaatg ttactagtca cacttgcact 4860
gtggatatct accgtgagat ggaagataaa gaatcacgga agttagtata ccatcccgcc 4920
actccggcgg ctggtcctct gcatggtgtg gcactgaata atccatatca gcctttgagt 4980
gtcattgatc tcaaacgctg ttctgctagg aataatagaa ctacatactg ctatgatttt 5040
ccactggcat ttgaaactgc agtgaggaag tcatggtcct ctagtacctc tggtgcttct 5100
aaaggtgttg aaaatgccca atgttatgtt aaagctacag agttggtatt tgcggacaaa 5160
catgggtcat ggggcactcc tttagttcaa atggaccggc ctgctgggct caatgacatt 5220
ggtatggtag cttggacctt gaagatgtcc actcctgaat ttcctagtgg tagggagatt 5280
attgttgttg caaatgatat tacgttcaga gctggatcat ttggcccaag ggaagatgca 5340
ttttttgaag ctgttaccaa cctagcctgt gagaagaaac ttcctcttat ttatttggca 5400
gcaaattctg gtgctcgaat tggcatagca gatgaagtga aatcttgctt ccgtgttggg 5460
tggtctgatg atggcagccc tgaacgtggg tttcagtaca tttatctaag cgaagaagac 5520
tatgctcgta ttggcacttc tgtcatagca cataagatgc agctagacag tggtgaaatt 5580
aggtgggtta ttgattctgt tgtgggcaag gaagatggac ttggtgtgga gaatatacat 5640
ggaagtgctg ctattgccag tgcttattct agggcatata aggagacatt tacacttaca 5700
tttgtgactg gaagaactgt tggaatagga gcttatcttg ctcgacttgg catccggtgc 5760
atacagcgtc ttgaccagta ctattattct tacaggctat tctgcactga acaagcttct 5820
tgggcgggaa gtgtacagct cccacatgca gttgggtggt cccaaaatca tggcaactaa 5880
tggtgttgtc catcttactg tttcagatga ccttgaaggc gtttctaata tattgaggtg 5940
gctcagttat gttcctgcct acattggtgg accacttcca gtaacaacac cgttggaccc 6000
accggacaga cctgttgcat acattcctga gaactcgtgt gatcctcgag cggctatccg 6060
tggtgttgat gacagccaag ggaaatggtt aggtggtatg tttgataaag acagctttgt 6120
ggaaacattt gaaggttggg ctaagacagt ggttactggc agagcaaagc ttggtggaat 6180
tccagtgggt gtgatagctg tggagactca gaccatgatg caaactatcc ctgctgaccc 6240
tggtcagctt gattcccgtg agcaatctgt tcctcgtgct ggacaagtgt ggtttccaga 6300
ttctgcaacc aagactgcgc aggcattgct ggacttcaac cgtgaaggat tacctctgtt 6360
catcctcgct aactggagag gcttctctgg tggacaaaga gatctttttg aaggaattct 6420
tcaggctggc tcgactattg ttgagaacct taggacatac aatcagcctg cctttgtcta 6480
cattcccatg gctgcagagc tacgaggagg ggcttgggtt gtggttgata gcaagataaa 6540
cccagaccgc attgagtgct atgctgagag gactgcaaaa ggcaatgttc tggaaccgca 6600
agggttaatt gagatcaagt tcaggtcaga ggaactccag gattgcatga gtcggcttga 6660
cccaacatta attgatctga aagcaaaact cgaagtagca aataaaaatg gaagtgctga 6720
cacaaaatcg cttcaagaaa atatagaagc tcgaacaaaa cagttgatgc ctctatatac 6780
tcagattgcg atacggtttg ctgaattgca tgatacatcc ctcagaatgg ctgcgaaagg 6840
tgtgattaag aaagttgtgg actgggaaga atcacgatct ttcttctata agagattacg 6900
gaggaggatc tctgaggatg ttcttgcaaa agaaattaga gctgtagcag gtgagcagtt 6960
ttcccaccaa ccagcaatcg agctgatcaa gaaatggtat tcagcttcac atgcagctga 7020
atgggatgat gacgatgctt ttgttgcttg gatggataac cctgaaaact acaaggatta 7080
tattcaatat cttaaggctc aaagagtatc ccaatccctc tcaagtcttt cagattccag 7140
ctcagatttg caagccctgc cacagggtct ttccatgtta ctagataaga tggatccctc 7200
tagaagagct caacttgttg aagaaatcag gaaggtcctt ggttga 7246
Claims (5)
1. A mutant gene of ACCase for resisting herbicide in plant,
the nucleotide sequence of the plant herbicide-resistant ACCase mutant gene consists of a sequence shown by SEQ ID NO. 1 in a sequence table.
2. An expression cassette comprising the ACCase mutant gene of claim 1.
3. A recombinant vector comprising the ACCase mutant gene of claim 1.
4. Use of the ACCase mutant gene of claim 1, the expression cassette of claim 2, and the recombinant vector of claim 3, wherein the use comprises introducing one of the ACCase mutant gene, the expression cassette, and the recombinant vector into a target rice to confer herbicide resistance to the target rice.
5. Use according to claim 4, wherein the gene is introduced into the recipient plant by means of transgenesis, crossing or backcrossing.
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